The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM

Abstract

Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery of Myxococcus xanthus. PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 of Pseudomonas aeruginosa formed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex.Type IV pili (T4P) 3 are versatile surface structures that are important for various processes, including twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are found on the surfaces of a wide variety of Gram-positive and Gram-negative bacteria and archaea (1-3). T4P systems (T4PS) are related to type II secretion systems (T2SS), which are responsible for the secretion of proteins across the outer membrane of Gram-negative bacteria (4, 5), bacterial competence systems that are involved in the uptake of DNA (6), and systems that are involved in the assembly of archaeal surface structures (7).T4P are highly dynamic structures that undergo cycles of extension and retraction (8, 9). During extensions, the T4P assembly ATPase PilB stimulates the extraction of pilin monomers from the inner membrane (IM) and their incorporation at the base of the pilus fiber. The fiber has a diameter of ϳ6 nm and can extend up to several micrometers in length (10). During retractions, the T4P disassembly ATPase PilT stimulates the removal of pilin monomers from the base of the pilus and their reinsertion into the IM (9, 11). T4P retraction generates forces up to 150 piconewtons (12, 13), pulling a cell forward, and making T4P systems the strongest molecular motors known.The rod-shaped cells of the ␦-proteobacterium Myxococcus xanthus, assemble 5-10 T4P at the leading cell pole that extend and retract to generate cell movemen...

PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM

“…Before manuscript publication, it was demonstrated that PilM directly interacts with PilB in Myxococcus xanthus (67), corroborating our results. Both BfpE, the PilC homologue in the T4bP system, and GspE, the PilB homologue in the T2SS, have been shown to bind to the PilM loop 1B equivalents in BfpC and GspL, respectively (24,51).…”

The molecular mechanism of the type IVa pilus motors

“…In this study it was hypothesised that PilB and PilT might function by rotating PilC to stimulate PilA polymerization or depolymerization9. In keeping with this model, PilC was recently shown to interact directly with and stimulate PilB activitiy10.…”

Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili

“…Using this methodology, multiple variations of pET29-based operons were generated, numbering up to seven pil genes. Toxicity/plasmid instability with pET29-based plasmids prompted us to subclone the above operons into the arabinose-inducible pBAD18 (32). Genes or groups of genes were extracted from pET-29 derivatives on an XbaI-XmaI fragment and subcloned into pBAD18 cut with NheI and XmaI.…”

“…but there is ample evidence that they establish multiple binary/ ternary interactions at the cytoplasmic membrane (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Moreover, in a recent study in Myxococcus xanthus, in which the entire Tfp machinery was visualized by cryoelectron tomography (34), it was shown that these five proteins form a series of interconnected layers spanning the cytoplasmic membrane, which is a priori compatible with a role in filament assembly.…”